Influence of Chlorinating Agents on the Formation of Stable Biomarkers in Hair for the Retrospective Verification of Exposure

Chlorine, as a dual-use chemical, is an essential industrial chemical which has been used as a chemical weapon in the past due to its toxicity and availability. The retrospective verification of chlorine intoxication is often especially challenging, and unambiguous markers are still missing. In this study, the effects of different chlorinating and oxidizing agents on human hair were investigated. Samples were exposed to a variety of chlorinating chemicals for a short time and then completely hydrolyzed by a HBr solution to break down their keratin proteins into individual amino acids. After derivatization and targeted liquid chromatography-mass spectrometry analysis, 3-chlorotyrosine and 3,5-dichlorotyrosine were unambiguously identified from human hair exposed to chlorine, hypochlorite, and sulfuryl chloride. Our results show long-term stability of these markers in the biological matrix, as the chlorotyrosines can still be found 10 months post-exposure at the same levels. Finally, an untargeted analysis was able to discriminate between some of the different intoxicants.


Exposure experiments
The exposures of hair samples to Cl 2 , HCl, phosgene, chloropicrin, oxalyl chloride, sulfuryl chloride and thionyl chloride were carried with the chlorinating chemicals being in the gas phase. Whereas the exposure to NaOCl and household bleach was carried out with the chemicals being in an aqueous solution (Table S 1).

S-3
Chlorine: The exposure experiments were carried out in an apparatus consisting of the Cl 2 cylinder with a pressure reducing valve, a first gas washing bottle (250 mL), the reaction vessel (100 mL two-necked round-bottomed flask) and a second gas washing station containing an aqueous 10 % NaOH solution to neutralize the Cl 2 .
The hair samples were placed in the reaction apparatus and Cl 2 (either concentrated, 100 ppm (+/-3% relative) in N 2 or 0.366 ppm (+/-10% relative) in N 2 were passed through the apparatus at room temperature for 10 minutes or 8 h, respectively. The remaining Cl 2 was removed by flushing with argon gas. The hair samples were collected and stored in Eppendorf tubes at room temperature under normal atmosphere (air).

NaOCl:
Hair samples were placed in aqueous NaOCl solution (1.5 mL) with concentrations of approx. 1'000 ppm ±10% and 10 ppm ±10% at room temperature for 10 minutes. The hair samples were collected and dried with a Kleenex.
Upon drying at room temperature for 2 h the samples were stored in Eppendorf tubes at room temperature.

Household bleach:
Hair samples were placed in commercially available household bleach solution (1.5 mL) at room temperature for 10 minutes. The hair samples were collected and dried with a Kleenex. Upon drying at room temperature for 2 h the samples were stored in Eppendorf tubes at room temperature.

HCl:
HCl was synthesized following a protocol by Arnáiz et al. 34 CaCl 2 (25 g, 225 mmol) was added to a 250 mL threenecked round-bottomed flask equipped with addition funnel and a gas outlet tube connected to the reaction chamber, a 100 mL two-necked round-bottomed flask. The gas was trapped using two gas washing tubes with deionized H 2 O (50 mL). The hair samples were placed into the reaction chamber and a solution of HCl (32%, 25 mL) was added dropwise over 10 minutes to the CaCl 2 forming white fumes. A constant stream of HCl could be observed in the gas washing tube. The hair samples were stored in Eppendorf tubes at room temperature.

Phosgene:
Phosgene (1 g, 10 mmol) was added to a 5'000 mL Schott flask. The flask was sealed with a stopper and Teflon tape. The flask was left stand at 19 °C for 19 h to let the phosgene evaporate. The phosgene did not evaporate completely. The flask was opened quickly, and the hair samples were placed according to Figure S 1. The hair samples were let in the flask for 10 minutes at 19 °C. The hair samples were removed and the upper part of the hair, which was not in contact with the vial (holder) was cut and transferred to Eppendorf tubes at room temperature. Chloropicrin: S-4 Chloropicrin (970 µL, 10 mmol) was added to a 5'000 mL Schott flask. The flask was sealed with a stopper and Teflon tape. The flask was left stand at 19 °C for 19 h to let the chloropicrin evaporate. The chloropicrin did not evaporate completely. The flask was opened quickly, and the hair samples were placed according to Figure S 1.
The hair samples were let in the flask for 10 minutes at 19 °C. The hair samples were removed and the upper part of the hair, which was not in contact with the vial (holder) was cut and transferred to Eppendorf tubes at room temperature.

S-5
Oxalyl chloride: Oxalyl chloride (858 µL, 10 mmol) was added to a 5'000 mL Schott flask. The flask was sealed with a stopper and Teflon tape. A white fog was formed at the bottom of the flask. It seemed that oxalyl chloride reacted with moisture in the flask and settled at the glass wall. After letting the liquid evaporate at 21 °C for 1 h, the flask was opened quickly, and the hair samples were placed according to Figure S 1. The hair samples were let in the flask for 10 minutes at 21 °C. The hair samples were removed and the upper part of the hair, which was not in contact with the vial (holder) was cut and transferred to Eppendorf tubes at room temperature.

Sulfuryl chloride:
Sulfuryl chloride (808 µL, 10 mmol) was added to a 5'000 mL Schott flask. The flask was sealed with a stopper and Teflon tape. After letting the liquid evaporate at 21 °C for 1 h, the flask was opened quickly, and the hair samples were placed according to Figure

MS parameters
The single quadrupole mass spectrometer was used with the following parameters. The cone voltage was set to 10 V for all amino acids and the probe temperature was set to 600°C. The voltage of electrospray was set to 0.6 KV.  The hydrolysate of the hair samples, as well as the amino acid reference standards, were derivatized with AQC. 37

Reference material & derivatization
A reaction scheme is depicted in Figure S 3. The amino acids asparagine, glutamine and tryptophan were not measured since asparagine and glutamine are converted into aspartic acid and glutamic acid respectively under the strong acidic conditions. Tryptophan is prone to degradation under acidic conditions and was therefore also not measured. 38 The compounds of interest were verified by the analysis of reference material ( Figure S 4 -8).    , TIC of Cl 2 exposed hair (red), extracted ion chromatogram (EIC) of 3-Cl-Tyr in the Cl 2 exposed hair sample (green) and the EIC of the 3-Cl-Tyr in the reference material sample (purple). Each chromatogram was normalized to the highest peak.
S-10  from a swimmer and Cl 2 conc. exposed hair for comparative purposes. A) 3-Cl-Tyr and B) 3,5-Cl 2 -Tyr only observed in significant amounts in hair exposed to concentrated Cl 2 . Untreated and Cl 2 exposed hair were measured in biological replicates, whereas the hair samples of the swimmer were measured in technical replicates. Exposure conditions are given in Table S 1. ns: not significant.